Immune responses occur in diverse anatomical sites, including protective responses to pathogens and dysregulated immune responses in autoimmune and inflammatory diseases. Mechanisms for the control of immune responses in vivo have been identified predominantly in mouse models, while our knowledge of human immune responses derives almost exclusively from studies of peripheral blood. This research program takes a new approach to address key deficiencies in our understanding of human immune responses, by focusing on immunity in human tissue sites. We have established a unique tissue resource involving the acquisition of multiple tissues from organ donors, through a collaboration with the New York Organ Donor Network (NYODN), giving us unprecedented access to all research-consented organ donors in the NY metropolitan area. The quality and quantity of the tissues, and our results showing reproducibility of data from diverse donors, has revealed an unprecedented potential of using this unique resource for groundbreaking studies in human immunology. The major goal of this research program is to take a novel, whole body approach to define how cells of the lymphoid lineage, are organized, function and adapted to tissue sites in steady state conditions and during an in situ response in intestinal transplantatio. We have assembled an outstanding team of top immunologists together with experts in bioinformatics and computational biology to use this unique tissue resource in conjunction with tissues from transplant recipients and employ multiple state-of-the-art approaches to investigate the cells, molecules and pathways involved in targeting immune responses to tissue sites. Project 1 (PI: D.L. Farber) will investigate the generation, diversity and homeostasis of T cell subsets throughout the body; Project 2 (PI: M.J. Shiomchik) will focus on B cell distribution, diversity and selection in tissues; Project 3 (PI: D. Artis) will analyze human innate lymphoid cells and regulation of tissue homeostasis; and Project 4 (P.I.: M. Sykes) will address lymphocyte reconstitution and responses in intestinal transplantation. All projects will obtain tissues and samples from a central human tissues core, and will be supported by an administrative/database management core and a bioinformatics core. The bioinformatics core will be essential for interpreting and coordinating the results from proposed analysis of immune diversity throughout the body, and gene expression analysis of tissue-resident subsets. Results from this research program will provide fundamental insights into human immunity in vivo and alter the way in which we interpret, treat and monitor disease. RELEVANCE: The research program will conduct novel studies of human immune cells in tissue sites throughout the body that have not previously been characterized, using a unique tissue resource set up by the Program PI. The results to be obtained and concepts to be learned will open a new frontier in human immunology and impact the way we analyze and interpret disease pathologies, design vaccines, and use immunotherapy to treat and cure intractable diseases. Project 1: Generation, Diversity and Homeostasis of T Cells Subsets throughout the Body Project 1 Leader (PL): Farber, Donna L. Description (as provided by applicant): Immune responses occur in diverse anatomical sites, including protective responses to pathogens and dysregulated immune responses in autoimmune and inflammatory diseases. However, in humans, our knowledge of T cell differentiation and maintenance in tissues remains sparse, and derives almost exclusively from studies of peripheral blood. My laboratory has carried out a unique whole body analysis of human T cells in lymphoid and mucosal tissues using tissue obtained from individual organ donors in collaboration with the New York Organ Donor Network (NYODN). Thus far, we have analyzed T cells from blood and eight different tissues including lymphoid tissues (spleen, peripheral and tissue-draining lymph nodes), and mucosal tissues (lung, small and large intestines) from over 24 research-consented, previously healthy organ donors. Our results reveal that the composition of nave, memory and terminal effector CD4 and CD8 subsets in blood, lymphoid and mucosal tissues is intrinsic to the tissue site and remarkably similar between diverse donors. Moreover, these subsets differentially express markers of activation and homeostasis in mucosal tissues, lymphoid tissues and blood, suggesting either in situ effects on T cell differentiation. Our central hypothesis is that compartmentalization of human T cells in tissues throughout the body depends on multiple mechanisms including the extent of replication, expression of specific T cell receptors (TCR), and tissue-specific factors. In the proposed research, we will 1. Evaluate the role of replication and turnover in tissue compartmentalization of T cell subsets, by examining whether different replication history and turnover is associated with residence in lymphoid versus mucosal sites; 2. Assess how TCR diversity and clonality is related to compartmentalization of T cell subsets in diverse anatomic sites using cutting edge deep sequencing approaches to directly probe whether tissue sites are enriched for specific T cell clones or contain limited TCR diversity compared to blood; and 3. Define the tissue-specific influences on T cells in circulating compared to lymphoid and mucosal sites through transcriptome analysis, by applying RNAseq analysis of T cell subsets in different sites to obtain novel tissue-specific gene signatures. Results from the proposed studies will provide valuable, new insights to how human immune responses are generated, maintained and regulated, essential for the development of new strategies for targeting tissue-specific immune responses in protective immunity, transplantation and autoimmunity. RELEVANCE: Translating findings in basic immunology from mouse to human is limited by our lack of fundamental knowledge of human T cells in tissue sites where they direct immune responses and are maintained. The proposed research will investigate how human T cells are compartmentalized on the cellular and molecular level in lymphoid and mucosal tissue sites using a unique tissue resource from organ donors. The results obtained will lead to new strategies for tissue targeting of T cell responses in vaccines and immunotherapies.